Methods for diagnosing cystic fibrosis

ABSTRACT

The present invention provides novel mutations of the CFTR gene related to cystic fibrosis or to conditions associated with cystic fibrosis. Also provided are probes for detecting the mutant sequences. Methods of identifying if an individual has a genotype containing one or more mutations in the CFTR gene are further provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/074,903, filed Mar. 7, 2005, which claims the benefit of U.S.Provisional Application No. 60,550,989, filed Mar. 5, 2004, incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel cystic fibrosis transmembraneregulator (CFTR) gene mutations and for detecting the presence of thesemutations in the CFTR gene of individuals.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Cystic fibrosis (CF) is the most common severe autosomal recessivegenetic disorder in the Caucasian population. It affects approximately 1in 2,500 live births in North America (Boat et al, The Metabolic Basisof Inherited Disease, 6th ed, pp 2649-2680, McGraw Hill, NY (1989)).Approximately 1 in 25 persons are carriers of the disease. Theresponsible gene has been localized to a 250,000 base pair genomicsequence present on the long arm of chromosome 7. This sequence encodesa membrane-associated protein called the “cystic fibrosis transmembraneregulator” (or “CFTR”). There are greater than 1000 different mutationsin the CFTR gene, having varying frequencies of occurrence in thepopulation, presently reported to the Cystic Fibrosis Genetic AnalysisConsortium. These mutations exist in both the coding regions (e.g.,ΔF508, a mutation found on about 70% of CF alleles, represents adeletion of a phenylalanine at residue 508) and the non-coding regions(e.g., the 5T, 7T, and 9T mutations correspond to a sequence of 5, 7, or9 thymidine bases located at the splice branch/acceptor site of intron8) of the CFTR gene.

The major symptoms of cystic fibrosis include chronic pulmonary disease,pancreatic exocrine insufficiency, and elevated sweat electrolytelevels. The symptoms are consistent with cystic fibrosis being anexocrine disorder. Although recent advances have been made in theanalysis of ion transport across the apical membrane of the epitheliumof CF patient cells, it is not clear that the abnormal regulation ofchloride channels represents the primary defect in the disease.

A variety of CFTR gene mutations are known. The identify of additionalmutations will further assist in the diagnosis of cystic fibrosis.

SUMMARY OF THE INVENTION

The inventors have discovered new mutations in the CFTR gene. Thesemutations, include 605G->C, 1198-1203del/1204G->A (deletes TGGGCT andreplaces G with A at position 1204), 1484G->T, 1573A->G, 1604G->C,1641-1642AG->T, 2949-2953del (deletes TACTC), 2978A->T, 3239C->A, and3429C->A, are related to the function of the CFTR gene and, therefore,to cystic fibrosis. These mutations are associated with cystic fibrosisor are associated with conditions associated with cystic fibrosis. By“conditions associated with cystic fibrosis” is meant any clinicalsymptoms that may be found in a cystic fibrosis patient and are due toone or more CF mutations.

Accordingly, in one aspect, the present invention provides a method ofdetermining if a CFTR gene contains one or more mutations selected fromthe group consisting of 605G->C, 1198-1203del/1204G->A (deletes TGGGCTand replaces G with A at position 1204), 1484G->T, 1573A->G, 1604G->C,1641-1642AG->T, 2949-2953del (deletes TACTC), 2978A->T, 3239C->A, and3429C->A, comprising determining if CFTR nucleic acid contains one ormore of the mutations.

In another aspect, the present invention provides a method ofidentifying if an individual has one or more mutations in the CFTR genecomprising determining if nucleic acid from the individual has one moremutations in one or both CFTR genes, the mutations selected from thegroup consisting of 605G->C, 1198-1203del/1204G->A (deletes TGGGCT andreplaces G with A at position 1204), 1484G->T, 1573A->G, 1604G->C,1641-1642AG->T, 2949-2953del (deletes TACTC), 2978A->T, 3239C->A, and3429C->A.

In yet another aspect, the present invention provides a method ofdetermining if an individual is predisposed to cystic fibrosis or to acondition associated with cystic fibrosis comprising determining ifnucleic acid from the individual has one more more mutations in one orboth CFTR genes, the mutations selected from the group consisting of605G->C, 1198-1203del/1204G->A (deletes TGGGCT and replaces G with A atposition 1204), 1484G->T, 1573A->G, 1604G->C, 1641-1642AG->T,2949-2953del (deletes TACTC), 2978A->T. 3239C->A, and 3429C->A.

In still a further aspect, the present invention provides a method ofcounseling an individual on the likelihood of having an offspringafflicted with cystic fibrosis or a condition associated with cysticfibrosis, comprising determining if nucleic acid from the individual hasone or more mutations in one or both CFTR genes, the mutations selectedfrom the group consisting of 605G->C, 1198-1203del/1204G->A (deletesTGGGCT and replaces G with A at position 1204), 1484G->T, 1573A->G,1604G->C, 1641-1642AG->T, 2949-2953del (deletes TACTC), 2978A->T,3239C->A, and 3429C->A.

In all of these aspects, the mutations may be 605G->C and 3239C->A. Insome embodiments, 1198-1203del (deletes TGGGCT) and the missense1204G->A may exist separately from the complex allele,1198-1203del/1204G->A (deletes TGGGCT and replaces G with A at position1204). In another embodiment, the mutations are selected from the groupconsisting of 1198-1203del/1204G->A (deletes TGGGCT and replaces G withA at position 1204), 1484G->T, 1604G->C, 1641-1642AG->T, 2949-2953del(deletes TACTC), 3239C->A, and 3429C->A. In another embodiment themutations are selected from the group consisting of 605G->C, 1573A->G,and 2978A->T.

In some embodiments, one more mutations are evaluated for both allelesof the CFTR gene in the individual. By this approach the genotype of theindividual can be determined at the position of each mutation.

The presence of the mutation in the CFTR gene may be determined by anyof a variety of well known methods used to detect single base changes(transitions and/or small deletions/insertions). Thus, genomic DNA maybe isolated from the individual and tested for the CF mutations. Inanother approach, mRNA can be isolated and tested for the CF mutations.Testing may be performed on mRNA or on a cDNA copy.

Genomic DNA or in cDNA may be subject to amplification by the polymerasechain reaction or related methods using primers directed to specificportions of the CFTR gene which contain a mutation to be detected. Thesequence of primers suitable for PCR amplification of portions of theCFTR gene in which contain the CF mutations are also provided.

The presence CF mutations can be determined in a nucleic acid bysequencing appropriate portions of the CFTR gene containing themutations sought to be detected. In another approach, CF mutations thatchange susceptibility to digestion by one or more endonucleaserestriction enzymes may be used to detect the mutations. In anotherembodiment, the presence of one or more CF mutations can be determinedby allele specific amplification. In yet another embodiment, thepresence of one or more CF mutations can be determined by primerextension. In yet a further embodiment, the presence of one or more CFmutations can be determined by oligonucleotide ligation. In anotherembodiment, the presence of one or more CF mutations can be determinedby hybridization with a detectably labeled probe containing the mutantCF sequence.

The methods of the invention also may include detection of other CFmutations which are known in the art and which are described herein.

The present invention also provides oligonucleotide probes that areuseful for detecting the CF mutations. Accordingly, provided is asubstantially purified nucleic acid comprising 8-20 nucleotides fullycomplementary to a segment of the CFTR gene that is fully complementaryto a portion of the CFTR gene and encompasses a mutant CFTR sequenceselected from the group consisting of 605G->C, 1204G->A, 1198-1203del(deletes TGGGCT), 1484G->T, 1573A->G, 1604G->C, 1641-1642AG->T,2949-2953del (deletes TACTC), 2978A->T, 3239C->A, and 3429C->A, or acomplementary nucleic acid sequence thereof. In one embodiment, thepurified nucleic acid is no more than 50 nucleotides in length. Theinvention CF mutant probes may be labeled with a detectable label, whichmay include any of a radioisotope, a dye, a fluorescent molecule, ahapten or a biotin molecule.

In another aspect the present invention provides kits for one of themethods described herein. In various embodiments, the kits contain oneor more of the following components in an amount sufficient to perform amethod on at least one sample: one or more primers of the presentinvention, one or more devices for performing the assay, which mayinclude one or more probes that hybridize to a mutant CF nucleic acidsequence, and optionally contain buffers, enzymes, and reagents forperforming a method of detecting a genotype of cystic fibrosis in anucleic acid sample.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a table showing various CFTR mutations and characterizinginformation.

DETAILED DESCRIPTION OF THE INVENTION

CF mutations and PCR primer pairs for amplifying segments of the CFTRgene containing the mutation are shown in Table 1.

TABLE 1 CF mutations and associated amplification primers Forward and CFMutation Nucleotide Change Reverse PCR Primers S158T 605G->C q4e1F andq4e1R V358I 1204G->A q7e3F and q7e4R 119del6 1198-1202del q7e3F andq7e4R (deletes TGGGCT and results in W356 and A357) G451V 1484G->T q9e9Fand q9e11R K481E 1573A->G s10e3F and s10e2R C491S 1604G->C s10e3F ands10e2R K503N + 1641-1642AG->T s10e3F and s10e2R frameshift (deletes1641A and 1642G and replaces with T) 2949del5 2949-2953del (deletesq15e3F and q15e4R TACTC) H949L 2978A->T q15e3F and q15e4R T1036N3239C->A q17ae1F and q17ae1R F1099L 3429C->A q17be1F and q17be1R

Further information relating to the CF mutations and the CFTR gene arefound in FIG. 1. The primers for amplifying segments of the CFTR genemay hybridize to coding or non-coding CFTR sequences under stringentconditions. Preferred primers are those that flank mutant CF sequences.Primers for CF mutations in Table 1 are shown in Table 2.

TABLE 2 Amplification primer sequences for CF mutations CF MutationForward and Reverse PCR Primers S158T q4e1F: (SEQ ID NO: 33)TGTAAAACGACGGCCAGTaaagtcttgtgttgaaattctcagg g4e1R: (SEQ ID NO: 34)CAGGAAACAGCTATGACCCAGCTCACTACCTAATTTATGACAT V3581 q7e3F: (SEQ ID NO: 35)119de16 TGTAAAACGACGGCCAGTcttccattccaagatccc g7e4R: (SEQ ID NO: 36)CAGGAAACAGCTATGACCGCAAAGTTCATTAGAACTGATC G451V g9e9F: (SEQ ID NO: 37)TGTAAAACGACGGCCAGTtggatcatgggccatgtgc and g9e11R: (SEQ ID NO: 38)CAGGAAACAGCTATGACCAAAGAGACATGGACACCAAATTAAG K481E s10e3F: (SEQ ID NO:39) C491S TGTAAAACGACGGCCAGTagcagagtacctgaaacagga K503N + s10e2R: (SEQID NO: 40) frameshift CAGGAAACAGCTATGACCCATTCACAGTAGCTTACCCA 2949del5q15e3F: (SEQ ID NO: 41) H949L TGTAAAACGACGGCCAGTggttaagggtgcatgctcttcq15e4R: (SEQ ID NO: 42) CAGGAAACAGCTATGACCGGCCCTATTGATGGTGGATC T1036Nq17ae1F: (SEQ ID NO: 43) TGTAAAACGACGGCCAGTacactttgtccactttgc q17ae1R:(SEQ ID NO: 44) CAGGAAACAGCTATGACCAGATGAGTATCGCACATTC F1099L q17be1F:(SEQ ID NO: 45) TGTAAAACGACGGCCAGTatctattcaaagaatggcac q17be1R: (SEQ IDNO: 46) CAGGAAACAGCTATGACCGATAACCTATAGAATGCAGC

By “mutations of the CFTR gene” or “mutant CF sequence” is meant one ormore CFTR nucleic acid sequences that are associated or correlated withcystic fibrosis. Additional CF mutations are disclosed in Table 3-17 maybe correlated with a carrier state, or with a person afflicted with CF.Thus, the nucleic acid may be tested for CF mutations described in anyof Tables 1-17. The nucleic acid sequences containing CF mutations arepreferably DNA sequences, and are preferably genomic DNA sequences;however, RNA sequences such as mRNA or hnRNA may also contain nucleicacid mutant sequences that are associated with cystic fibrosis.

By “carrier state” is meant a person who contains one CFTR allele thatis a mutant CF nucleic acid sequence, but a second allele that is not amutant CF nucleic acid sequence. CF is an “autosomal recessive” disease,meaning that a mutation produces little or no phenotypic effect whenpresent in a heterozygous condition with a non-disease related allele,but produces a “disease state” when a person is homozygous, i.e., bothCFTR alleles are mutant CF nucleic acid sequences.

By “primer” is meant a sequence of nucleic acid, preferably DNA, thathybridizes to a substantially complementary target sequence and isrecognized by DNA polymerase to begin DNA replication.

By “substantially complementary” is meant that two sequences hybridizeunder stringent hybridization conditions. The skilled artisan willunderstand that substantially complementary sequences need not hybridizealong their entire length. In particular, substantially complementarysequences comprise a contiguous sequence of bases that do not hybridizeto a target sequence, positioned 3′ or 5′ to a contiguous sequence ofbases that hybridize under stringent hybridization conditions to atarget sequence.

By “flanking” is meant that a primer hybridizes to a target nucleic acidadjoining a region of interest sought to be amplified on the target. Theskilled artisan will understand that preferred primers are pairs ofprimers that hybridize 3′ from a region of interest, one on each strandof a target double stranded DNA molecule, such that nucleotides may beadd to the 3′ end of the primer by a suitable DNA polymerase. Primersthat flank mutant CF sequences do not actually anneal to the mutantsequence but rather anneal to sequence that adjoins the mutant sequence.

By “isolated” a nucleic acid (e.g., an RNA, DNA or a mixed polymer) isone which is substantially separated from other cellular componentswhich naturally accompany such nucleic acid. The term embraces a nucleicacid sequence which has been removed from its naturally occurringenvironment, and includes recombinant or cloned DNA isolates,oligonucleotides, and chemically synthesized analogs or analogsbiologically synthesized by heterologous systems.

By “substantially pure” a nucleic acid, represents more than 50% of thenucleic acid in a sample. The nucleic acid sample may exist in solutionor as a dry preparation.

By “complement” is meant the complementary sequence to a nucleic acidaccording to standard Watson/Crick pairing rules. For example, asequence (SEQ ID NO: 1) 5′-GCGGTCCCAAAAG-3′ has the complement (SEQ IDNO: 2) 5′-CTTTTGGGACCGC-3′. A complement sequence can also be a sequenceof RNA complementary to the DNA sequence or its complement sequence, andcan also be a cDNA.

By “coding sequence” is meant a sequence of a nucleic acid or itscomplement, or a part thereof, that can be transcribed and/or translatedto produce the mRNA for and/or the polypeptide or a fragment thereof.Coding sequences include exons in a genomic DNA or immature primary RNAtranscripts, which are joined together by the cell's biochemicalmachinery to provide a mature mRNA. The anti-sense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

By “non-coding sequence” is meant a sequence of a nucleic acid or itscomplement, or a part thereof, that is not transcribed into amino acidin vivo, or where tRNA does not interact to place or attempt to place anamino acid. Non-coding sequences include both intron sequences ingenomic DNA or immature primary RNA transcripts, and gene-associatedsequences such as promoters, enhancers, silencers, etc.

Nucleic acid suspected of containing mutant CF sequences are amplifiedusing one or more primers that flank the mutations under conditions suchthat the primers will amplify CFTR fragments containing the mutations,if present. The oligonucleotide sequences in Table 2 are useful foramplifying segments of the CFTR gene which contain the mutations inTable 1. Nucleic acid from an individual also could be tested for CFTRmutations other than those in Table 1. Such mutations include, forexample, any of those listed in Tables 4-18. Primers for these latterCFTR mutations include

The method of identifying the presence or absence of mutant CF sequenceby amplification can be used to determine whether a subject has agenotype containing one or more nucleotide sequences correlated withcystic fibrosis. The presence of a wildtype or mutant sequence at eachpredetermined location can be ascertained by the invention methods.

By “amplification” is meant one or more methods known in the art forcopying a target nucleic acid, thereby increasing the number of copiesof a selected nucleic acid sequence. Amplification may be exponential orlinear. A target nucleic acid may be either DNA or RNA. The sequencesamplified in this manner form an “amplicon.” While the exemplary methodsdescribed hereinafter relate to amplification using the polymerase chainreaction (“PCR”), numerous other methods are known in the art foramplification of nucleic acids (e.g., isothermal methods, rolling circlemethods, etc.). The skilled artisan will understand that these othermethods may be used either in place of, or together with, PCR methods.

The nucleic acid suspected of containing mutant CF sequence may beobtained from a biological sample. By “biological sample” is meant asample obtained from a biological source. A biological sample can, byway of non-limiting example, consist of or comprise blood, sera, urine,feces, epidermal sample, skin sample, cheek swab, sperm, amniotic fluid,cultured cells, bone marrow sample and/or chorionic villi. Convenientbiological samples may be obtained by, for example, scraping cells fromthe surface of the buccal cavity. The term biological sample includessamples which have been processed to release or otherwise make availablea nucleic acid for detection as described herein. For example, abiological sample may include a cDNA that has been obtained by reversetranscription of RNA from cells in a biological sample.

By “subject” is meant a human or any other animal which contains as CFTRgene that can be amplified using the primers and methods describedherein. A subject can be a patient, which refers to a human presentingto a medical provider for diagnosis or treatment of a disease. A humanincludes pre and post natal forms. Particularly preferred subjects arehumans being tested for the existence of a CF carrier state or diseasestate.

By “identifying” with respect to an amplified sample is meant that thepresence or absence of a particular nucleic acid amplification productis detected. Numerous methods for detecting the results of a nucleicacid amplification method are known to those of skill in the art.

The present invention provides specific primers that aid in thedetection of mutant CF genotype. Such primers enable the amplificationof segments of the CFTR gene that are known to contain mutant CFsequence from a nucleic acid containing biological sample. By amplifyingspecific regions of the CFTR gene, the invention primers facilitate theidentification of wildtype or mutant CF sequence at a particularlocation of the CFTR gene. Primers for amplifying various regions of theCFTR gene include the following: SEQ ID NO: 3.5′-GCGGTCCCAAAAGGGTCAGTTGTAGGAAGTCACCAAAG-3′ (g4e1F), and SEQ ID NO:4,5′-GCGGTCCCAAAAGGGTCAGTCGATACAGAATATATGTGCC-3′ (g4e2R), are preferablyused together as forward (F) and reverse (R) primers; SEQ ID NO:5,5′-GCGGTCCCAAAAGGGTCAGTGAATCATTCAGTGGGTATAAGCAG-3′ (g19i2F), and SEQID NO: 6,5′-GCGGTCCCAAAAGGGTCAGTCTTCAATGCACCTCCTCCC-3′ (q19i3R), arepreferably used together as forward (F) and reverse (R) primers; SEQ IDNO: 7, 5′-GCGGTCCCAAAAGGGTCAGTAGATACTTCAATAGCTCAGCC-3′ (g7e1F), and SEQID NO: 8,5′-GCGGTCCCAAAAGGGTCAGTGGTACATTACCTGTATTTTGTTT-3′ (g7e2R), arepreferably used together as forward (F) and reverse (R) primers; SEQ IDNO: 9, 5′-GCGGTCCCAAAAGGGTCAGTGTGAATCGATGTGGTGACCA-3′ (s12e1F), and SEQID NO: 10, 5′-GCGGTCCCAAAAGGGTCAGTCTGGTTTAGCATGAGGCGGT-3′ (s12e1R), arepreferably used together as forward (F) and reverse (R) primers; SEQ IDNO: 11, 5′-GCGGTCCCAAAAGGGTCAGTTTGGTTGTGCTGTGGCTCCT-3′ (g14be1F), andSEQ ID NO: 12, 5′-GCGGTCCCAAAAGGGTCAGTACAATACATACAAACATAGTGG-3′(g14be2R), are preferably used together as forward (F) and reverse (R)primers; SEQ ID NO: 13,5′-GCGGTCCCAAAAGGGTCAGTGAAAGTATTTATTTTTTCTGGAAC-3′ (q21e1F), and SEQ IDNO: 14 5′-GCGGTCCCAAAAGGGTCAGTGTGTGTAGAATGATGTCAGCTAT-3′ (q21e2R), arepreferably used together as forward (F) and reverse (R) primers; SEQ IDNO: 15, 5′-GCGGTCCCAAAAGGGTCAGTCAGATTGAGCATACTAAAAGTG-3′ (g11e1F), andSEQ ID NO: 16, 5′-GCGGTCCCAAAAGGGTCAGTTACATGAATGACATTTACAGCA-3′(g11e2R), are preferably used together as forward (F) and reverse (R)primers; SEQ ID NO: 17, 5′-GCGGTCCCAAAAGGGTCAGTAAGAACTGGATCAGGGAAGA-3′(g20e1F), and SEQ ID NO: 18,5′-GCGGTCCCAAAAGGGTCAGTTCCTTTTGCTCACCTGTGGT-3′ (g20e2R), are preferablyused together as forward (F) and reverse (R) primers; SEQ ID NO: 19,5′-GCGGTCCCAAAAGGGTCAGTGGTCCCACTTTTTATTCTTTTGC-3′ (q3e2F), and SEQ IDNO: 20 5′-GCGGTCCCAAAAGGGTCAGTTGGTTTCTTAGTGTTTGGAGTTG-3′ (q3e2R), arepreferably used together as forward (F) and reverse (R) primers; SEQ IDNO: 21, 5′-GCGGTCCCAAAAGGGTCAGTTGGATCATGGGCCATGTGC-3′ (g9e9F), and SEQID NO: 22, 5′-GCGGTCCCAAAAGGGTCAGTACTACCTTGCCTGCTCCAGTGG-3′ (g9e9R), arepreferably used together as forward (F) and reverse (R) primers; SEQ IDNO: 23, 5′-GCGGTCCCAAAAGGGTCAGTAGGTAGCAGCTATTTTTATGG-3′ (g13e2F), andSEQ ID NO: 24, 5′-GCGGTCCCAAAAGGGTCAGTTAAGGGAGTCTTTTGCACAA-3′ (g13e2R),are preferably used together as forward (F) and reverse (R) primers; SEQID NO: 25

5′-GCGGTCCCAAAAGGGTCAGTGCAATTTTGGATGACCTTC-3′ (q16i1F), and SEQ ID NO:26 5′-GCGGTCCCAAAAGGGTCAGTTAGACAGGACTTCAACCCTC-3′ (q16i2R), arepreferably used together as forward (F) and reverse (R) primers; SEQ IDNO: 27, 5′-GCGGTCCCAAAAGGGTCAGTGGTGATTATGGGAGAACTGG-3′ (q10e10F), andSEQ ID NO: 28, 5′-GCGGTCCCAAAAGGGTCAGTATGCTTTGATGACGCTTC-3′ (q10e11R),are preferably used together as forward (F) and reverse (R) primers; SEQID NO: 29, 5′-GCGGTCCCAAAAGGGTCAGTTTCATTGAAAAGCCCGAC-3′ (q19e12F), andSEQ ID NO: 30, 5′-GCGGTCCCAAAAGGGTCAGTCACCTTCTGTGTATTTTGCTG-3′ (q19e13R)are preferably used together as forward (F) and reverse (R) primers; andSEQ ID NO: 31, 5′-GCGGTCCCAAAAGGGTCAGTAAGTATTGGACAACTTGTTAGTCTC-3′(q5e12F), and SEQ ID NO: 32,5′-GCGGTCCCAAAAGGGTCAGTCGCCTTTCCAGTTGTATAATTT-3′ (q5e13R), arepreferably used together as forward (F) and reverse (R) primers. Thesepairs of primers, which may been used in multiplex amplifications, canamplify the regions of the CFTR gene shown in Table 3.

TABLE 3 CFTR Primer Pairs and Amplicon Characteristics Exon/ ForwardPrimer Reverse Primer Intron Size g14be1F (SEQ ID NO. 11) g14be24 (SEQID NO. 12) 14b/ 149 i14b q5e12F (SEQ ID NO. 31) q5e13R (SEQ ID NO. 32)5/i5 165 g20e1F (SEQ ID NO. 17) g20e2R (SEQ ID NO. 18) 20 194 q16i1F(SEQ ID NO. 25) q16i2R (SEQ ID NO. 26) 16/i16 200 q10e10F (SEQ ID NO.27) q10e11R (SEQ ID NO. 28) 10 204 q21e1F (SEQ ID NO. 13) q21e2R (SEQ IDNO. 14) 21 215 g11e1F (SEQ ID NO. 15) g11e2R (SEQ ID NO. 16) i10/11/ 240i11 g7e1F (SEQ ID NO. 7) g7e2R (SEQ ID NO. 8)  7 259 g4e1F (SEQ ID NO.3) g4e2R (SEQ ID NO. 4) 4/i4 306 q3e2F (SEQ ID NO. 19) q3e2R (SEQ ID NO.20) 3/i3 308 q19e12F (SEQ ID NO. 29) q1913e2R i18/19 310 (SEQ ID NO. 30)q13e2F (SEQ ID NO. 23) g13e2R (SEQ ID NO. 24) 13 334 g9e9F (SEQ ID NO.21) g9e9R (SEQ ID NO. 22) i8/9 351 g19i2F (SEQ ID NO. 5) g19i3R (SEQ IDNO. 6) i19 389 s12e1F (SEQ ID NO. 9) s12e1R (SEQ ID NO. 10) i11/12/ 465i12

The nucleic acid to be amplified may be from a biological sample such asan organism, cell culture, tissue sample, and the like. The biologicalsample can be from a subject which includes any eukaryotic organism oranimal, preferably fungi, invertebrates, insects, arachnids, fish,amphibians, reptiles, birds, marsupials and mammals. A preferred subjectis a human, which may be a patient presenting to a medical provider fordiagnosis or treatment of a disease. The biological sample may beobtained from a stage of life such as a fetus, young adult, adult, andthe like. Particularly preferred subjects are humans being tested forthe existence of a CF carrier state or disease state.

The sample to be analyzed may consist of or comprise blood, sera, urine,feces, epidermal sample, skin sample, cheek swab, sperm, amniotic fluid,cultured cells, bone marrow sample and/or chorionic villi, and the like.A biological sample may be processed to release or otherwise makeavailable a nucleic acid for detection as described herein. Suchprocessing may include steps of nucleic acid manipulation, e.g.,preparing a cDNA by reverse transcription of RNA from the biologicalsample. Thus, the nucleic acid to be amplified by the methods of theinvention may be DNA or RNA.

Nucleic acid may be amplified by one or more methods known in the artfor copying a target nucleic acid, thereby increasing the number ofcopies of a selected nucleic acid sequence. Amplification may beexponential or linear. The sequences amplified in this manner form an“amplicon.” In a preferred embodiment, the amplification by the is bythe polymerase chain reaction (“PCR”) (e.g., Mullis, K. et al., ColdSpring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al.,European Patent Appln. 50,424; European Patent Appln. 84,796, EuropeanPatent Application 258,017, European Patent Appln. 237,362; Mullis, K.,European Patent Appln. 201,184; Mullis K. et al., U.S. Pat. No.4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al.,U.S. Pat. No. 4,683,194). Other known nucleic acid amplificationprocedures that can be used include, for example, transcription-basedamplification systems or isothermal amplification methods (Malek, L. T.et al., U.S. Pat. No. 5,130,238; Davey, C. et al. European PatentApplication 329,822; Schuster et al., U.S. Pat. No. 5,169,766; Miller,H. I. et al., PCT appln. WO 89/06700; Kwoh, D. et al., Proc. Natl. Acad.Sci. (U.S.A.) 86:1173 (1989); Gingeras, T. R. et al., PCT application WO88/10315; Walker, G. T. et al., Proc. Natl. Acad. Sci. (U.S.A.)89:392-396 (1992)). Amplification may be performed to with relativelysimilar levels of each primer of a primer pair to generate an doublestranded amplicon. However, asymmetric PCR may be used to amplifypredominantly or exclusively a single stranded product as is well knownin the art (e.g., Poddar et al. Molec. And Cell. Probes 14:25-32(2000)). This can be achieved for each pair of primers by reducing theconcentration of one primer significantly relative to the other primerof the pair (e.g. 100 fold difference). Amplification by asymmetric PCRis generally linear. One of ordinary skill in the art would know thatthere are many other useful methods that can be employed to amplifynucleic acid with the invention primers (e.g., isothermal methods,rolling circle methods, etc.), and that such methods may be used eitherin place of, or together with, PCR methods. Persons of ordinary skill inthe art also will readily acknowledge that enzymes and reagentsnecessary for amplifying nucleic acid sequences through the polymerasechain reaction, and techniques and procedures for performing PCR, arewell known. The examples below illustrate a standard protocol forperforming PCR and the amplification of nucleic acid sequences thatcorrelate with or are indicative of cystic fibrosis.

In another aspect, the present invention provides methods of detecting acystic fibrosis genotype in a biological sample. The methods compriseamplifying nucleic acids in a biological sample of the subject andidentifying the presence or absence of one or more mutant cysticfibrosis nucleic acid sequences in the amplified nucleic acid.Accordingly, the present invention provides a method of determining thepresence or absence of one or more mutant cystic fibrosis nucleic acidsequences in a nucleic acid containing sample, comprising: contactingthe sample with reagents suitable for nucleic acid amplificationincluding one or more pairs of nucleic acid primers flanking one or morepredetermined nucleic acid sequences that are correlated with cysticfibrosis, amplifying the predetermined nucleic acid sequence(s), ifpresent, to provide an amplified sample; and identifying the presence orabsence of mutant or wildtype sequences in the amplified sample.

One may analyze the amplified product for the presence of absence of anyof a number of mutant CF sequences that may be present in the samplenucleic acid. As already discussed, numerous mutations in the CFTR genehave been associated with CF carrier and disease states. For example, athree base pair deletion leading to the omission of a phenylalanineresidue in the gene product has been determined to correspond to themutations of the CF gene in approximately 70% of the patients affectedby CF. The table below identifies preferred CF sequences and identifieswhich of the primer pairs of the invention may be used to amplify thesequence.

TABLE 4 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 19 and 20. Name Nucleotide change ExonConsequence 297 − 3C −> T C to T at 297 − 3 intron 2 mRNA splicingdefect? E56K G to A at 298 3 Glu to Lys at 56 300delA Deletion of A at300 3 Frameshift W57R T to C at 301 3 Trp to Arg at 57 W57G T to G at301 3 Trp to Gly at 57 W57X(TAG) G to A at 302 3 Trp to Stop at 57W57X(TGA) G to A at 303 3 Trp to Stop at 57 D58N G to A at 304 3 Asp toAsn at 58 D58G A to G at 305 3 Asp to Gly at 58 306insA Insertion of A 3Frameshift at 306 306delTAGA deletion of TAGA 3 Frameshift from 306 E60LG to A at 310 3 Glu to Leu at 60 E60X G to T at 310 3 Glu to Stop at 60E60K G to A at 310 3 Glu to Lys at 60 N66S A to G at 328 3 Asn to Ser at66 P67L C to T at 332 3 Pro to Leu at 67 P68E A to G at 334 3 Lys to Gluat 68 K68N A to T at 336 3 Lys to Asn at 68 A72T G to A at 346 3 Ala toThr at 72 A72D C to A at 347 3 Ala to Asp at 72 347delC deletion of C at347 3 Frameshift R74W C to T at 352 3 Arg to Trp at 74 R74Q G to A at353 3 Arg to Gln at 74 R75X C to T at 355 3 Arg to Stop at 75 R75L G toT at 356 3 Arg to Leu at 75 359insT Insertion of T 3 Frameshift after359 360delT deletion of T at 360 3 Frameshift W79R T to C at 367 3 Trpto Arg at 79 W79X G to A at 368 3 Trp to Stop at 79 G85E G to A at 386 3Gly to Glu at 85 G85V G to T at 386 3 Gly to Val at 85 F87L T to C at391 3 Phe to Leu at 87 394delTT deletion of TT 3 frameshift from 394L88S T to C at 395 3 Leu to Ser at 88 L88X(T −> A) T to A at 395 3 Leuto Stop at 88 L88X(T −> G) T to G at 395 3 Leu to Stop at 88 Y89C A to Gat 398 3 Tyr to Cys at 89 L90S T to C at 401 3 Leu to Ser at 90 G91R Gto A at 403 3 Gly to Arg at 91 405 + 1G −> A G to A at 405 + 1 intron 3mRNA splicing defect 405 + 3A −> C A to C at 405 + 3 intron 3 mRNAsplicing defect? 405 + 4A −> G A to G at 405 + 4 intron 3 mRNA splicingdefect?

TABLE 5 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 3 and 4. Name Nucleotide change ExonConsequence A96E C to A at 419 4 Ala to Glu at 96 Q98X C to T at 424 4Gln to Stop at 98 (Pakistani specific?) Q98P A to C at 425 4 Gln to Proat 98 Q98R A to G at 425 4 Gln to Arg at 98 P99L C to T at 428 4 Pro toLeu at 99 L101X T to G at 434 4 Leu to Stop at 101 435insA Insertion ofA 4 Frameshift after 435 G103X G to T at 439 4 Gly to Stop at 103441delA deletion of A at 441 4 Frameshift and T to A at 486 444delAdeletion of A at 444 4 Frameshift I105N T to A at 446 4 Ile to Asn at105 451del8 deletion of 4 Frameshift GCTTCCTA from 451 S108F C to T at455 4 Ser to Phe at 108 457TAT −> G TAT to G at 457 4 Frameshift Y109N Tto A at 457 4 Tyr to Asn at 109 458delAT deletion of AT 4 Frameshift at458 Y109C A to G at 458 4 Tyr to Cys at 109 460delG deletion of G at 4604 Frameshift D110Y G to T at 460 4 Asp to Tyr at 110 D110H G to C at 4604 Asp to His at 110 D110E C to A at 462 4 Asp to Glu at 110 P111A C to Gat 463 4 Pro to Ala at 111 P111L C to T at 464 4 Pro to Leu at 111[delta]E115 3 bp deletion of 4 deletion of Glu at 115 475-477 E116Q G toC at 478 4 Glu to Gln at 116 E116K G to A at 478 4 Glu to Lys at 116R117C C to T at 481 4 Arg to Cys at 117 R117P G to C at 482 4 Arg to Proat 117 R117L G to T at 482 4 Arg to Leu at 117 R117H G to A at 482 4 Argto His at 117 I119V A to G at 487 4 Iso to Val at 119 A120T G to A at490 4 Ala to Thr at 120 Y122X T to A at 498 4 Tyr to Stop at 122 I125T Tto C at 506 4 Ile to Thr at 125 G126D G to A at 509 4 Gly to Asp at 126L127X T to G at 512 4 Leu to Stop at 127 525delT deletion of T at 525 4Frameshift 541del4 deletion of 4 Frameshift CTCC from 541 541delCdeletion of C at 541 4 Frameshift L137R T to G at 542 4 Leu to Arg at137 L137H T to A at 542 4 Leu to His at 137 L138ins insertion of CTA, 4insertion of leucine at TAC or ACT at 138 nucleotide 544, 545 or 546546insCTA Insertion of CTA 4 Frameshift at 546 547insTA insertion of TA4 Frameshift after 547 H139L A to T at 548 4 His to Leu at 548 H139R Ato G at 548 4 His to Arg at 139 P140S C to T at 550 4 Pro to Ser at 140P140L C to T at 551 4 Pro to Leu at 140 552insA Insertion of A 4Frameshift after 552 A141D C to A at 554 4 Ala to Asp at 141 556delAdeletion of A at 556 4 Frameshift 557delT deletion of T at 557 4Frameshift 565delC deletion of C at 565 4 Frameshift H146R A to G at 5694 His to Arg at 146 (CBAVD) 574delA deletion of A at 574 4 FrameshiftI148N T to A at 575 4 Ile to Asn at 148 I148T T to C at 575 4 Ile to Thrat 148 G149R G to A at 577 4 Gly to Arg at 149 Q151X C to T at 583 4 Glnto Stop at 151 M152V A to G at 586 4 Met to Val at 152 (mutation?) M152RT to G at 587 4 Met to Arg at 152 591del18 deletion of 18 bp 4 deletionof 6 amino acids from 591 from the CFTR protein A155P G to C at 595 4Ala to Pro at 155 S158R A to C at 604 4 Ser to Arg at 158 605insTInsertion of T 4 Frameshift after 605 L159X T to A at 608 4 Leu to Stopat 159 Y161D T to G at 613 4 Tyr to Asp at 161 Y161N T to A at 613 4 Tyrto Asn at 161 Y161S A to C at 614 4 Tyr to Ser at 161 (together with612T/A) K162E A to G at 616 4 Lys to Glu at 162 621G −> A G to A at 6214 mRNA splicing defect 621 + 1G −> T G to T at 621 + 1 intron 4 mRNAsplicing defect 621 + 2T −> C T to C at 621 + 2 intron 4 mRNA splicingdefect 621 + 2T −> G T to G at 621 + 2 intron 4 mRNA splicing defect621 + 3A −> G A to G at 621 + 3 intron 4 mRNA splicing defect

TABLE 6 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 31 and 32. Name Nucleotide_change ExonConsequence 681delC deletion of C at 681 5 Frameshift N186K C to A at690 5 Asn to Lys at 186 N187K C to A at 693 5 Asn to Lys at 187[delta]D192 deletion of TGA or 5 deletion of Asp at 192 GAT from 706 or707 D192N G to A at 706 5 Asp to Asn at 192 D192G A to G at 707 5 Asp toGly at 192 E193K G to A at 709 5 Glu to Lys at 193 E193X G to T at 709 5Glu to Stop at 193 711 + 1G −> T G to T at 711 + 1 intron mRNA splicingdefect 5 711 + 3A −> G A to G at 711 + 3 intron mRNA splicing defect 5711 + 3A −> C A to C at 711 + 3 intron mRNA splicing defect 5 711 + 3A−> T A to T at 711 + 3 intron mRNA splicing defect? 5 711 + 5G −> A G toA at 711 + 5 intron mRNA splicing defect 5 711 + 34A −> G A to G at711 + 34 intron mRNA splicing defect? 5

TABLE 7 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 7 and 8. Name Nucleotide_change ExonConsequence [delta]F311 deletion of 3 bp 7 deletion of Phe 310, between1059 and 311 or 312 1069 F311L C to G at 1065 7 Phe to Leu at 311 G314RG to C at 1072 7 Gly to Arg at 314 G314E G to A at 1073 7 Gly to Glu at314 G314V G to T at 1073 7 Gly to Val at 324 F316L T to G at 1077 7 Pheto Leu at 316 1078delT deletion of T 7 Frameshift at 1078 V317A T to Cat 1082 7 Val to Ala at 317 L320V T to G at 1090 7 Leu to Val at 320CAVD L320X T to A at 1091 7 Leu to Stop at 320 L320F A to T at 1092 7Leu to Phe at 320 V322A T to C at 1097 7 Val to Ala at 322 (mutation?)1112delT deletion of T 7 Frameshift at 1112 L327R T to G at 1112 7 Leuto Arg at 327 1119delA deletion of A 7 Frameshift at 1119 G330X G to Tat 1120 7 Gly to Stop at 330 R334W C to T at 1132 7 Arg to Trp at 334R334Q G to A at 1133 7 Arg to Gln at 334 R334L G to T at 1133 7 Arg toLeu at 334 1138insG Insertion of G after 7 Frameshift 1138 I336K T to Aat 1139 7 Ile to Lys at 336 T338I C to T at 1145 7 Thr to Ile at 3351150delA deletion of A at 7 Frameshift 1150 1154insTC insertion of TCafter 7 Frameshift 1154 1161insG Insertion of G after 7 Frameshift 11611161delC deletion of C at 7 Frameshift 1161 L346P T to C at 1169 7 Leuto Pro at 346 R347C C to T at 1171 7 Arg to Cys at 347 R347H G to A at1172 7 Arg to His at 347 R347L G to T at 1172 7 Arg to Leu at 347 R347PG to C at 1172 7 Arg to Pro at 347 M348K T to A at 1175 7 Met to Lys at348 A349V C to T at 1178 7 Ala to Val at 349 R352W C to T at 1186 7 Argto Trp at 352 R352Q G to A at 1187 7 Arg to Gln at 352 Q353X C to T at1189 7 Gln to Stp at 353 Q353H A to C at 1191 7 Gln to His at 3531199delG deletion of G at 7 Frameshift 1199 W356X G to A at 1200 7 Trpto Stop at 356 Q359K/T360K C to A at 1207 and 7 Glu to Lys at 359 and Cto A at 1211 Thr to Lys at 360 Q359R A to G at 1208 7 Gln to Arg at 3591213delT deletion of T at 7 Frameshift 1213 W361R(T −> C) T to C at 12137 Trp to Arg at 361 W361R(T −> A) T to A at 1213 7 Trp to Arg at 3611215delG deletion of G at 7 Frameshift 1215 1221delCT deletion of CT 7Frameshift from 1221 S364P T to C at 1222 7 Ser to Pro at 364 L365P T toC at 1226 7 Leu to Pro at 365

TABLE 8 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 21 and 22. Name Nucleotide_change ExonConsequence 1342 − 11TTT −> G TTT to G at 1342 − 11 intron mRNA splicing8 defect? 1342 − 2delAG deletion of AG from intron Frameshift 1342 − 2 81342 − 2A −> C A to C at 1342 − 2 intron mRNA splicing 8 defect 1342 −1G −> C G to C at 1342 − 1 intron mRNA splicing 8 defect E407V A to T at1352 9 Glu to Val at 407 1366delG deletion of G at 1366 9 Frameshift1367delC deletion of C at 1367 9 Frameshift 1367del5 deletion of CAAAAat 9 Frameshift 1367 Q414X C to T at 1372 9 Gln to Stop at 414 N418S Ato G at 1385 9 Asn to Ser at 418 G424S G to A at 1402 9 Gly to Ser at424 S434X C to G at 1433 9 Ser to Stop at 434 D443Y G to T at 1459 9 Aspto Tyr at 443 1460delAT deletion of AT from 9 Frameshift 1460 1461ins4insertion of AGAT after 9 Frameshift 1461 I444S T to G at 1463 9 Ile toSer at 444 1471delA deletion of A at 1471 9 Frameshift Q452P A to C at1487 9 Gln to Pro at 452 [delta]L453 deletion of 3 bp between 9 deletionof Leu 1488 and 1494 at 452 or 454 A455E C to A at 1496 9 Ala to Glu at455 V456F G to T at 1498 9 Val to Phe at 456

TABLE 9 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 27 and 28. Name Nucleotide_change ExonConsequence G480C G to T at 1570 10 Gly to Cys at 480 G480D G to A at1570 10 Gly to Asp at 480 G480S G to A at 1570 10 Gly to Ser at 4801571delG deletion of G at 1571 10 Frameshift 1576insT Insertion of T at1576 10 Frameshift H484Y C to T at 1582 10 His to Tyr at 484 (CBAVD?)H484R A to G at 1583 10 His to Arg at 484 S485C A to T at 1585 10 Ser toCys at 485 G486X G to T at 1588 10 Glu to Stop at 486 S489X C to A at1598 10 Ser to Stop at 489 1601delTC deletion of TC from 1601 or 10Frameshift CT from 1602 C491R T to C at 1603 10 Cys to Arg at 491 S492FC to T at 1607 10 Ser to Phe at 492 Q493X C to T at 1609 10 Gln to Stopat 493 1609delCA deletion of CA from 1609 10 Frameshift Q493R A to G at1610 10 Gln to Arg at 493 1612delTT deletion of TT from 1612 10Frameshift W496X G to A at 1619 10 Trp to Stop at 496 P499A C to G at1627 10 Pro to Ala at 499 (CBAVD) T501A A to G at 1633 10 Thr to Ala at501 I502T T to C at 1637 10 Ile to Thr at 502 I502N T to A at 1637 10Ile to Asn at 502 E504X G to T at 1642 10 Glu to Stop at 504 E504Q G toC at 1642 10 Glu to Gln at 504 I506L A to C at 1648 10 Ile to Leu at 506[delta]I507 deletion of 3 bp between 1648 10 deletion of Ile506 or and1653 Ile507 I506S T to G at 1649 10 Ile to Ser at 506 I506T T to C at1649 10 Ile to Thr at 506 [delta]F508 deletion of 3 bp between 1652 10deletion of Phe at and 1655 508 F508S T to C at 1655 10 Phe to Ser at508 D513G A to G at 1670 10 Asp to Gly at 513 (CBAVD) 1677delTA deletionof TA from 1677 10 Frameshift Y517C A to G at 1682 10 Tyr to Cys at 517

TABLE 10 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 15 and 16. Name Nucleotide_change ExonConsequence 1716 − 1G −> A G to A at 1716 − 1 intron mRNA splicing 10defect 1717 − 8G −> A G to A at 1717 − 8 intron mRNA splicing 10 defect?1717 − 3T −> G T to G at 1717 − 3 intron mRNA splicing 10 defect? 1717 −2A −> G A to G at 1717 − 2 intron mRNA splicing 10 defect 1717 − 1G −> AG to A at 1717 − 1 intron mRNA splicing 10 defect D529H G to C at 171711 Asp to His at 529 1717 − 9T −> A T to A at 1717 − 9 intron mRNAsplicing 10 mutation? A534E C to A at 1733 11 Ala to Glu at 5341742delAC deletion of AC from 11 Frameshift 1742 I539T T to C at 1748 11Ile to Thr at 539 1749insTA Insertion of TA 11 frameshift at 1749resulting in premature termination at 540 G542X G to T at 1756 11 Gly toStop at 542 G544S G to A at 1762 11 Gly to Ser at 544 G544V G to T at1763 11 Gly to Val at 544 (CBAVD) 1774delCT deletion of CT from 11Frameshift 1774 S549R(A −> C) A to C at 1777 11 Ser to Arg at 549 S549IG to T at 1778 11 Ser to Ile at 549 S549N G to A at 1778 11 Ser to Asnat 549 S549R(T −> G) T to G at 1779 11 Ser to Arg at 549 G550X G to T at1780 11 Gly to Stop at 550 G550R G to A at 1780 11 Gly to Arg at 5501782delA deletion of A at 11 Frameshift 1782 G551S G to A at 1783 11 Glyto Ser at 551 1784delG deletion of G at 11 Frameshift 1784 G551D G to Aat 1784 11 Gly to Asp at 551 Q552X C to T at 1786 11 Gln to Stop at 552Q552K C to A at 1786 11 Gln to Lys 1787delA deletion of A at 11frameshift, stop position 1787 or codon at 558 1788 R553G C to G at 178911 Arg to Gly at 553 R553X C to T at 1789 11 Arg to Stop at 553 R553Q Gto A at 1790 11 Arg to Gln at 553 (associated with [delta]F508; R555G Ato G at 1795 11 Arg to Gly at 555 I556V A to G at 1798 11 Ile to Val at556 (mutation?) 1802delC deletion of C at 11 Frameshift 1802 L558S T toC at 1805 11 Leu to Ser at 558 1806delA deletion of A at 11 Frameshift1806 A559T G to A at 1807 11 Ala to Thr at 559 A559E C to A at 1808 11Ala to Glu at 559 A560T G to C at 1811 11 Arg to Thr at 560; mRNAsplicing defect? R560K G to A at 1811 11 Arg to Lys at 560 1811 + 1G −>C G to C at 1811 + 1 intron mRNA splicing 11 defect 1811 + 1.6 kb A −> GA to G at intron creation of splice 1811 + 1.2 kb 11 donor site 1811 +18G −> A G to A at 1811 + 18 intron mRNA splicing 11 defect?

TABLE 11 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 9 and 10. Name Nucleotide change ExonConsequence 1812 − 1G −> A G to A at 1812 − 1 intron mRNA splicingdefect 11 R560S A to C at 1812 12 Arg to Ser at 560 1813insC Insertionof C after 12 Frameshift 1813 (or 1814) A561E C to A at 1814 12 Ala toGlu at 561 V562I G to A at 1816 12 Val to Ile at 562 V562L G to C at1816 12 Val to Leu at 562 Y563D T to G at 1819 12 Tyr to Asp at 563Y563N T to A at 1819 12 Tyr to Asn at 563 Y563C A to G at 1821 12 Tyr toCys at 563 1833delT deletion of T at 12 Frameshift 1833 L568X T to A at1835 12 Leu to Stop at 568 L568F G to T at 1836 12 Leu to Phe at 568(CBAVD?) Y569D T to G at 1837 12 Tyr to Asp at 569 Y569H T to C at 183712 Tyr to His at 569 Y569C A to G at 1838 12 Tyr to Cys at 569 V569X Tto A at 1839 12 Tyr to Stop at 569 L571S T to C at 1844 12 Leu to Ser at571 1845delAG/ deletion of AG at 12 Frameshift 1846delGA 1845 or GA at1846 D572N G to A at 1846 12 Asp to Asn at 572 P574H C to A at 1853 12Pro to His at 574 G576X G to T at 1858 12 Gly to Stop at 576 G576A G toC at 1859 12 Gly to Ala at 576 (CAVD) Y577F A to T at 1862 12 Tyr to Pheat 577 D579Y G to T at 1867 12 Asp to Tyr at 579 D579G A to G at 1868 12Asp to Gly at 579 D579A A to C at 1868 12 Asp to Ala at 579 1870delGdeletion of G at 12 Frameshift 1870 1874insT Insertion of T 12Frameshift between 1871 and 1874 T582R C to G at 1877 12 Thr to Arg at582 T582I C to T at 1877 12 Thr to Ile at 582 E585X G to T at 1885 12Glu to Stop at 585 S589N G to A at 1898 12 Ser to Asn at 589 (mRNAsplicing defect?) S589I G to T at 1898 12 Ser to Ile at 589 (splicing?)1898 + 1G −> A G to A at 1898 + 1 intron mRNA splicing defect 12 1898 +1G −> C G to C at 1898 + 1 intron mRNA splicing defect 12 1898 + 1G −> TG to T at 1898 + 1 intron mRNA splicing defect 12 1898 + 3A −> G A to Gat 1898 + 3 intron mRNA splicing defect? 12 1898 + 3A −> C A to C at1898 + 3 intron mRNA splicing defect? 12 1898 + 5G −> A G to A at 1898 +5 intron mRNA splicing defect 12 1898 + 5G −> T G to T at 1898 + 5intron mRNA splicing defect 12 1898 + 73T −> G T to G at 1898 + 73intron mRNA splicing defect? 12

TABLE 12 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 23 and 24. Name Nucleotide_change ExonConsequence 1918delGC deletion of GC from 1918 13 Frameshift 1924del7deletion of 7 bp 13 Frameshift (AAACTA) from 1924 R600G A to G at 193013 Arg to Gly at 600 I601F A to T at 1933 13 Ile to Phe at 601 V603F Gto T at 1939 13 Val to Phe at 603 T604I C to T at 1943 13 Thr to Ile at604 1949del84 deletion of 84 bp from 1949 13 deletion of 28 a.a. (Met607to Gln634) H609R A to G at 1958 13 His to Arg at 609 L610S T to C at1961 13 Leu to Ser at 610 A613T G to A at 1969 13 Ala to Thr at 613D614Y G to T at 1972 13 Asp to Tyr 614 D614G A to G at 1973 13 Asp toGly at 614 I618T T to C at 1985 13 Ile to Thr at 618 L619S T to C at1988 13 Leu to Ser at 619 H620P A to C at 1991 13 His to Pro at 620H620Q T to G at 1992 13 His to Gln at 620 G622D G to A at 1997 13 Gly toAsp at 622 (oligospermia) G628R(G −> A) G to A at 2014 13 Gly to Arg at628 G628R(G −> C) G to C at 2014 13 Gly to Arg at 628 L633P T to C at2030 13 Leu to Pro at 633 Q634X T to A at 2032 13 Gln to Stop at 634L636P T to C at 2039 13 Leu to Pro at 636 Q637X C to T at 2041 13 Gln toStop at 637 2043delG deletion of G at 2043 13 Frameshift 2051delTTdeletion of TT from 2051 13 Frameshift 2055del9 −> A deletion of 9 bp 13Frameshift CTCAAAACT to A at 2055 D648V A to T at 2075 13 Asp to Val at648 D651N G to A at 2083 13 Asp to Asn at 651 E656X T to G at 2098 13Glu to Stop at 656 2108delA deletion of A at 2108 13 Frameshift 2109del9−> A deletion of 9 bp from 2109 13 Frameshift and insertion of A2113delA deletion of A at 2113 13 Frameshift 2116delCTAA deletion ofCTAA at 2116 13 Frameshift 2118del4 deletion of AACT 13 Frameshift from2118 E664X G to T at 2122 13 Glu to Stop at 664 T665S A to T at 2125 13Thr to Ser at 665 2141insA Insertion of A after 2141 13 Frameshift2143delT deletion of T at 2143 13 Frameshift E672del deletion of 3 bpbetween 13 deletion of Glu at 2145-2148 672 G673X G to T at 2149 13 Glyto Stop at 673 W679X G to A at 2168 13 Trp to stop at 679 2176insCInsertion of C after 2176 13 Frameshift K683R A to G at 2180 13 Lys toArg at 683 2183AA −> G A to G at 2183 and deletion 13 Frameshift of A at2184 2183delAA deletion of AA at 2183 13 Frameshift 2184delA deletion ofA at 2184 13 frameshift 2184insG insertion of G after 2184 13 Frameshift2184insA Insertion of A after 2184 13 Frameshift 2185insC Insertion of Cat 2185 13 Frameshift Q685X C to T at 2185 13 Gln to Stop at 685 E692X Gto T at 2206 13 Glu to Stop at 692 F693L(CTT) T to C at 2209 13 Phe toLeu at 693 F693L(TTG) T to G at 2211 13 Phe to Leu at 693 2215insGInsertion of G at 2215 13 Frameshift K698R A to G 2225 13 Lys to Arg at698 R709X C to T at 2257 13 Arg to Stop at 709 K710X A to T at 2260 13Lys to Stop at 710 K716X AA to GT at 2277 and 2278 13 Lys to Stop at 716L719X T to A at 2288 13 Leu to Stop at 719 Q720X C to T at 2290 13 Glnto stop codon at 720 E725K G to A at 2305 13 Glu to Lys at 725 2307insAInsertion of A after 2307 13 Frameshift E730X G to T at 2320 13 Glu toStop at 730 L732X T to G at 2327 13 Leu to Stop at 732 2335delA deletionof A at 2335 13 Frameshift R735K G to A at 2336 13 Arg to Lys at 7352347delG deletion of G at 2347 13 Frameshift 2372del8 deletion of 8 bpfrom 2372 13 Frameshift P750L C to T at 2381 13 Pro to Leu at 750 V754MG to A at 2392 13 Val to Met at 754 T760M C to T at 2411 13 Thr to Metat 760 R764X C to T at 2422 13 Arg to Stop at 764 2423delG deletion of Gat 2423 13 Frameshift R766M G to T at 2429 13 Arg to Met at 7662456delAC deletion of AC at 2456 13 Frameshift S776X C to G at 2459 13Ser to Stop at 776

TABLE 13 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 11 and 12. Name Nucleotide_change ExonConsequence T908N C to A at 2788 14b Thr to Asn at 908 2789 + 2insAinsertion of A intron mRNA splicing defect? after 2789 + 2 14b (CAVD)2789 + 3delG deletion of G at intron mRNA splicing defect 2789 + 3 14b2789 + 5G −> A G to A at 2789 + 5 intron mRNA splicing defect 14b

TABLE 14 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 25 and 26. Name Nucleotide_change ExonConsequence 3100insA Insertion of A after 16 Frameshift 3100 I991V A toG at 3103 16 Ile to Val at 991 D993Y G to T at 3109 16 Asp to Tyr at 993F994C T to G at 3113 16 Phe to Cys at 994 3120G −> A G to A at 3120 16mRNA splicing defect 3120 + 1G −> A G to A at 3120 + 1 intron mRNAsplicing defect 16

TABLE 15 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 29 and 30. Name Nucleotide_change ExonConsequence 3601 − 20T −> C T to C at 3601 − 20 intron mRNA splicingmutant? 18 3601 − 17T −> C T to C at 3601 − 17 intron mRNA splicingdefect? 18 3601 − 2A −> G A to G at 3601 − 2 intron mRNA splicing defect18 R1158X C to T at 3604 19 Arg to Stop at 1158 S1159P T to C at 3607 19Ser to Pro at 115p S1159F C to T at 3608 19 Ser to Phe at 1159 R1162X Cto T at 3616 19 Arg to Stop at 1162 3622insT Insertion of T after 19Frameshift 3622 D1168G A to G at 3635 19 Asp to Gly at 1168 3659delCdeletion of C at 19 Frameshift 3659 K1177X A to T at 3661 19 Lys to Stpat 3661 (premature termination) K1177R A to G at 3662 19 Lys to Arg at1177 3662delA deletion of A at 19 Frameshift 3662 3667del4 deletion of 4bp 19 Frameshift from 3667 3667ins4 insertion of TCAA 19 Frameshiftafter 3667 3670delA deletion of A at 19 Frameshift 3670 Y1182X C to G at3678 19 Tyr to Stop at 1182 Q1186X C to T at 3688 19 Gln to Stop codonat 1186 3696G/A G to A at 3696 18 No change to Ser at 1188 V1190P T to Aat 3701 19 Val to Pro at 1190 S1196T C or Q at 3719 19 Ser-Top at 1196S1196X C to G at 3719 19 Ser to Stop at 1196 3724delG deletion of G at19 Frameshift 3724 3732delA deletion of A at 19 Frameshift and Lys to3732 and A to Glu at 1200 G at 3730 3737delA deletion of A at 19Frameshift 3737 W1204X G to A at 3743 19 Trp to Stop at 1204 S1206X C toG at 3749 19 Ser to Stop at 1206 3750delAG deletion of AG from 19Frameshift 3750 3755delG deletion of G 19 Frameshift between 3751 and3755 M1210I G to A at 3762 19 Met to Ile at 1210 M1212I G to A at 376619 Val to Ile at 1212

TABLE 16 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 5 and 6. Name Nucleotide_change ExonConsequence 3849 + 10 kb C −> T C to T in a 6.2 kb intron creation ofsplice EcoRI fragment 19 acceptor site 10 kb from 19

TABLE 17 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 17 and 18. Name Nucleotide_change ExonConsequence T1252P A to C at 3886 20 Thr to Pro at 1252 L1254X T to G at3893 20 Leu to Stop at 1254 S1255P T to C at 3895 20 Ser to Pro at 1255S1255L C to T at 3896 20 Ser to Leu at 1255 S1255X C to A at 3896 and 20Ser to Stop at 1255 and A to G at 3739 in Ile to Val at 1203 exon 193898insC Insertion of C after 20 Frameshift 3898 F1257L T to G at 390320 Phe to Leu at 1257 3905insT Insertion of T after 20 Frameshift 39053906insG Insertion of G after 20 Frameshift 3906 [delta]L1260 deletionof ACT 20 deletion of Leu at 1260 from either 3909 or 1261 or 39123922del10 −> C deletion of 10 bp 20 deletion of Glu1264 to from 3922 andGlu1266 replacement with 3921 I1269N T to A at 3938 20 Ile to Asn at1269 D1270N G to A at 3940 20 Asp to Asn at 1270 3944delGT deletion ofGT from 20 Frameshift 3944 W1274X G to A at 3954 20 Trp to Stop at 1274Q1281X C to T at 3973 20 Gln to Stop at 1281 W1282R T to C at 3976 20Trp to Arg at 1282 W1282G T to G at 3976 20 Trp to Gly at 1282 W1282X Gto A at 3978 20 Trp to Stop at 1282 W1282C G to T at 3978 20 Trp to Cysat 1282 R1283M G to T at 3980 20 Arg to Met at 1283 R1283K G to A at3980 20 Arg to Lys at 1283 F1286S T to C at 3989 20 Phe to Ser at 1286

TABLE 18 CFTR mutations that may be detected in amplified product usingas the primer pair SEQ ID NO: 13 and 14. Name Nucleotide_change ExonConsequence T1299I C to T at 4028 21 Thr to Ile at 1299 F1300L T to C at4030 21 Phe to Leu at 1300 N1303H A to C at 4039 21 Asn to His at 1303N1303I A to T at 4040 21 Asn to Ile at 1303 4040delA deletion of A at4040 21 Frameshift N1303K C to G at 4041 21 Asn to Lys at 1303 D1305E Tto A at 4047 21 Asp to Glu at 1305 4048insCC insertion of CC after 404821 Frameshift Y1307X T to A at 4053 21 Tyr to Stop at 1307 E1308X G to Tat 4054 21 Glu to Stop at 1308

CF mutations including those known under symbols: 2789+5G>A; 711+1G>T;W1282X; 3120+1G>A; d1507; dF508; (F508C, 1507V, 1506V); N₁₃O₃K; G542X,G551D, R553X, R560T, 1717-1G>A: R334W, R347P, 1078delT; R117H, 1148T,621+1G>T; G85E; R1162X, 3659delC; 2184delA; A455E, (5T, 7T, 9T); 3849+10kbC>T; and 1898+1G>A, are described in U.S. patent application Ser. No.396,894, filed Apr. 22, 1989, application Ser. No. 399,945, filed Aug.29, 1989, application Ser. No. 401,609 filed Aug. 31, 1989. and U.S.Pat. Nos. 6,001,588 and 5,981,178, which are hereby incorporated byreference in their entirety. Any and all of these mutations can bedetected using nucleic acid amplified with the invention primers asdescribed herein.

CF mutations in the amplified nucleic acid may be identified in any of avariety of ways well known to those of ordinary skill in the art. Forexample, if an amplification product is of a characteristic size, theproduct may be detected by examination of an electrophoretic gel for aband at a precise location. In another embodiment, probe molecules thathybridize to the mutant or wildtype CF sequences can be used fordetecting such sequences in the amplified product by solution phase or,more preferably, solid phase hybridization. Solid phase hybridizationcan be achieved, for example, by attaching the CF probes to a microchip.Probes for detecting CF mutant sequences are well known in the art. SeeWall et al. “A 31-mutation assay for cystic fibrosis testing in theclinical molecular diagnostics laboratory,” Human Mutation, 1995;5(4):333-8, which specifies probes for CF mutations ΔF508 (exon 1),G542X (exon 11), G551D (exon 11), R117H (exon 4), W1282X (exon 20),N₁₃O₃K (exon 21), 3905insT (exon 20), 3849+10 Kb (intron 19), G85E (exon3), R334W (exon 7), A455E (exon 9), 1898+1 (exon 12), 2184delA (exon13), 711+1 (exon 5), 2789+5 (exon 14b), Y1092x exon 17b), Δ1507 (exon10), S549R(T-G) (exon 11), 621+1 (exon 4), R1162X (exon 19), 1717-1(exon 11), 3659delC (exon 19), R560T (exon 11), 3849+4 (A-G) (exon 19),Y122X (exon 4), R553X (exon 11), R347P (exon 7), R347H (exon 7), Q493X(exon 10), V520F (exon 10), and S549N (exon 11).

CF probes for detecting mutations as described herein may be attached toa solid phase in the form of an array as is well known in the art (see,U.S. Pat. Nos. 6,403,320 and 6,406,844). For example, the fullcomplement of 24 probes for CF mutations with additional control probes(30 in total) can be conjugated to a silicon chip essentially asdescribed by Jenison et al., Biosens Bioelectron. 16(9-12):757-63 (2001)(see also U.S. Pat. Nos. 6,355,429 and 5,955,377). Amplicons thathybridized to particular probes on the chip can be identified bytransformation into molecular thin films. This can be achieved bycontacting the chip with an anti-biotin antibody or streptavidinconjugated to an enzyme such as horseradish peroxidase. Followingbinding of the antibody (or streptavidin)-enzyme conjugate to the chip,and washing away excess unbound conjugate, a substrate can be added suchas tetramethylbenzidine (TMB) {3,3′,5,5′Tetramethylbenzidine} to achievelocalized deposition (at the site of bound antibody) of a chemicalprecipitate as a thin film on the surface of the chip. Otherenzyme/substrate systems that can be used are well known in the art andinclude, for example, the enzyme alkaline phosphatase and5-bromo-4-chloro-3-indolyl phosphate as the substrate. The presence ofdeposited substrate on the chip at the locations in the array whereprobes are attached can be read by an optical scanner. U.S. Pat. Nos.6,355,429 and 5,955,377, which are hereby incorporated by reference intheir entirety including all charts and drawings, describe preferreddevices for performing the methods of the present invention and theirpreparation, and describes methods for using them.

The binding of amplified nucleic acid to the probes on the solid phasefollowing hybridization may be measured by methods well known in the artincluding, for example, optical detection methods described in U.S. Pat.No. 6,355,429. In preferred embodiments, an array platform (see, e.g.,U.S. Pat. No. 6,288,220) can be used to perform the methods of thepresent invention, so that multiple mutant DNA sequences can be screenedsimultaneously. The array is preferably made of silicon, but can beother substances such as glass, metals, or other suitable material, towhich one or more capture probes are attached. In preferred embodiments,at least one capture probe for each possible amplified product isattached to an array. Preferably an array contains 10, more preferably20, even more preferably 30, and most preferably at least 60 differentcapture probes covalently attached to the array, each capture probehybridizing to a different CF mutant sequence. Nucleic acid probesuseful as positive and negative controls also may be included on thesolid phase or used as controls for solution phase hybridization.

Another approach, variously referred to as PCR amplification of specificallele (PASA) (Sarkar, et al., 1990 Anal. Biochem. 186:64-68),allele-specific amplification (ASA) (Okayama, et al., 1989 J. Lab. Clin.Med. 114:105-113), allele-specific PCR (ASPCR) (Wu, et al. 1989 Proc.Natl. Acad. Sci. USA. 86:2757-2760), and amplification-refractorymutation system (ARMS) (Newton, et al., 1989 Nucleic Acids Res.17:2503-2516). The method is applicable for single base substitutions aswell as micro deletions/insertions. In general, two complementaryreactions are used. One contains a primer specific for the normal alleleand the other reaction contains a primer for the mutant allele (bothhave a common 2nd primer). One PCR primer perfectly matches one allelicvariant of the target, but is mismatched to the other. The mismatch islocated at/near the 3′ end of the primer leading to preferentialamplification of the perfectly matched allele. Genotyping is based onwhether there is amplification in one or in both reactions. A band inthe normal reaction only indicates a normal allele. A band in the mutantreaction only indicates a mutant allele. Bands in both reactionsindicate a heterozygote. As used herein, this approach will be referredto as “allele specific amplification.”

In yet another approach, restriction fragment length polymorphism(RFLP), which refers to the digestion pattern of various restrictionenzymes applied to DNA. RFLP analysis can be applied to PCR amplifiedDNA to identify CF mutations as disclosed herein.

In still another approach, wildtype or mutant CF sequence in amplifiedDNA may be detected by direct sequence analysis of the amplifiedproducts. A variety of methods can be used for direct sequence analysisas is well known in the art. See, e.g., The PCR Technique: DNASequencing (eds. James Ellingboe and Ulf Gyllensten) BiotechniquesPress, 1992; see also “SCAIP” (single condition amplification/internalprimer) sequencing, by Flanigan et al. Am J Hum Genet. 2003 April;72(4):931-9. Epub 2003 Mar. 11. Direct sequencing of CF mutations isalso described in Strom et al., 2003 Genetics in Medicine 5(1):9-14.

In yet another approach for detecting wildtype or mutant CF sequences inamplified DNA is single nucleotide primer extension or “SNuPE.” SNuPEcan be performed as described in U.S. Pat. No. 5,888,819 to Goelet etal., U.S. Pat. No. 5,846,710 to Bajaj, Piggee, C. et al. Journal ofChromatography A 781 (1997), p. 367-375 (“Capillary Electrophoresis forthe Detection of Known Point Mutations by Single-Nucleotide PrimerExtension and Laser-Induced Fluorescence Detection”); Hoogendoorn, B. etal., Human Genetics (1999) 104:89-93, (“Genotyping Single NucleotidePolymorphism by Primer Extension and High Performance LiquidChromatography”); and U.S. Pat. No. 5,885,775 to Haff et al. (analysisof single nucleotide polymorphism analysis by mass spectrometry). InSNuPE, one may use as primers such as those specified in Table 17.

Another method for detecting CF mutations include the Luminex xMAPsystem which has been adapted for cystic fibrosis mutation detection byTM Bioscience and is sold commercially as a universal bead array(Tag-It™).

Still another approach for detecting wildtype or mutant CF sequences inamplified DNA is oligonucleotide ligation assay or “OLA” or “OL”. TheOLA uses two oligonucleotides which are designed to be capable ofhybridizing to abutting sequences of a single strand of a targetmolecules. One of the oligonucleotides is biotinylated, and the other isdetectably labeled. If the precise complementary sequence is found in atarget molecule, the oligonucleotides will hybridize such that theirtermini abut, and create a ligation substrate that can be captured anddetected. See e.g., Nickerson et al. (1990) Proc. Natl. Acad. Sci.U.S.A. 87:8923-8927, Landegren, U. et al. (1988) Science 241:1077-1080and U.S. Pat. No. 4,998,617.

These above approaches for detecting wildtype or mutant CF sequence inthe amplified nucleic acid is not meant to be limiting, and those ofskill in the art will understand that numerous methods are known fordetermining the presence or absence of a particular nucleic acidamplification product.

In another aspect the present invention provides kits for one of themethods described herein. The kit optionally contain buffers, enzymes,and reagents for amplifying the CFTR nucleic acid via primer-directedamplification. The kit also may include one or more devices fordetecting the presence or absence of particular mutant CF sequences inthe amplified nucleic acid. Such devices may include one or more probesthat hybridize to a mutant CF nucleic acid sequence, which may beattached to a bio-chip device, such as any of those described in U.S.Pat. No. 6,355,429. The bio-chip device optionally has at least onecapture probe attached to a surface on the bio-chip that hybridizes to amutant CF sequence. In preferred embodiments the bio-chip containsmultiple probes, and most preferably contains at least one probe for amutant CF sequence which, if present, would be amplified by a set offlanking primers. For example, if five pairs of flanking primers areused for amplification, the device would contain at least one CF mutantprobe for each amplified product, or at least five probes. The kit alsopreferably contains instructions for using the components of the kit.

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention

EXAMPLES Example 1 Sample Collection and Preparation

Whole Blood: 5 cc of whole blood is collected in a lavender-top (EDTA)tube or yellow-top (ACD) tube. Green-top (Na Heparin) tubes areacceptable but less desirable. DNA is extracted from blood. 100 ng ormore DNA is prepared in TE or sterile water.

Amniotic Fluid: 10-15 cc of Amniotic Fluid is collected in a sterileplastic container.

Cultured Cells: Two T-25 culture flasks with 80-100% confluent growthmay be used.

Chorionic 10-20 mg of Chorionic Villi are collected in a sterilecontainer. 2-3 mL of sterile saline or tissue culture medium is added.

Transport: Whole Blood, Amniotic Fluid, Cultured Cells and ChorionicVilli can be shipped at room temperature (18°-26° C.). Amniotic Fluid,Cultured Cells or Chorionic Villi preferably is used withoutrefrigeration or freezing. Whole Blood and Extracted DNA can be shippedat 2°-10° C.

Storage: Whole Blood, Amniotic Fluid and Extracted DNA are stored at2°-10° C., Cultured Cells and Chorionic Villi are stored at roomtemperature (18°-26° C.).

Stability: Whole Blood is generally stable for 8 days at roomtemperature (18°-26° C.) or 8 days refrigerated at 2°-10° C. AmnioticFluid, Cultured Cells, and Chorionic Villi are generally processed toobtain DNA within 24 hours of receipt. Extracted DNA is stable for atleast 1 year at 2°-10° C.

Example 2 Amplification from DNA

Polymerase chain reaction (PCR) primer pairs were designed using theCFTR gene sequences in EMBL/Genbank (Accession Nos. M55106-M55131). EachPCR primer for the 32 separate PCR reactions contains either an M13forward linker sequence or an M13 reverse linker sequence as appropriateto allow universal sequence reaction priming. Individual PCR reactionsare performed in 96-well microtiter plates under the same conditions foreach amplicon. Subsequently, the PCR products are purified with theMillipore Montage™ PCR₉₆ Cleanup kit (Millipore, Bedford, Mass.) on aBeckman BioMek 2,000 biorobot. Further details are provided in Strom etal., 2003 Genetics in Medicine 5(1):9-14.

In general, individual amplifications are prepared in a volume of 13.5μl, which is added to the 96 well microtiter plates. Each amplificationvolume contained 2 μl of the nucleic acid sample (generally 10-100 ng ofDNA), 11.5 μl of PCR-Enzyme Mix (PCR-Enzyme mix stock is prepared with11.3 μl master mix, 0.25 μl MgCl₂ (from 25 mM stock), and 0.2 μl ofFasStar Taq (source for last two reagents was Roche Applied science,Cat. No. 2 032 937). Master mix contained primers, Roche PCR buffer withMgCl₂, Roche GC rich solution (cat. No. 2 032 937), bovine serum albumin(BSA) (New England BioLabs, Cat no. B9001B), and NTPs (AmershamBiosciences, Cat no. 27-2032-01).

The final concentration in the PCR for MgCl₂ was 2.859 mM, for BSA is0.725 μg/μl, and for each dNTP is 0.362 mM. Primer final concentrationsvaried from about 0.29 μM to about 0.036 μM

PCR is conducted using the following temperature profile: step 1: 96° C.for 15 minutes; step 2: 94° C. for 15 seconds; step 3: decrease at 0.5°C./second to 56° C.; step 4: 56° C. for 20 seconds; step 5: increase at0.3° C./second to 72° C., step 6: 72° C. for 30 seconds; step 7:increase 0.5° C. up to 94° C.; step 8: repeat steps 2 to 7 thirty threetimes; step 9: 72° C. for 5 minutes; step 10: 4° C. hold (to stop thereaction).

Example 3 Detection of CF Mutations

The purified PCR products are diluted to approximately 10 ng/μL andcycle sequencing reactions are performed with an ABI Prism Big Dye™Terminator v3.0 cycle sequencing reaction kit (Applied Biosystems,Foster City, Calif.) according to the manufacturer's protocol. The DNAprimers used for the sequencing reaction are M13 forward and reverseprimers as appropriate. Big Dye™ Terminator reaction products arepurified by the Millipore Montage™ Seq₉₆Sequencing Reaction Cleanup kiton a biorobot and analyzed on an ABI Prism 3100 Genetic Analyzer.Sequences obtained are examined for the presence of mutations by usingABI SeqScape v1.1 software. Both strands of DNA are sequenced.

All PCR reactions, purifications, and cycle sequencing reactions areperformed in 96-well microtiter plates using biorobots to avoid errorsintroduced by manual setups. Loading of samples onto the capillarysequencer is also automated. One plate is sufficient to perform theentire sequencing reaction for a single patient. Theoretically, if allreactions were successful, the entire sequences for a single patientcould be obtained in 24-48 hours after receipt of blood. In practice,however, one or more reactions must be repeated because of frequentpolymorphisms in intron 8 and 6a and failed reactions.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

Applicants reserve the right to physically incorporate into thisapplication any and all materials and information from any sucharticles, patents, patent applications, or other physical and electronicdocuments.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. Other embodimentsare within the following claims. In addition, where features or aspectsof the invention are described in terms of Markush groups, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup.

1. A method of determining the cystic fibrosis status of a human comprising: (a) determining the presence or absence of the 1484G->T mutation in both alleles of the cystic fibrosis transmembrane regulatory (CFTR) gene in a nucleic acid sample obtained from the human, and (b) identifying the human (i) as having cystic fibrosis or being predisposed to cystic fibrosis when the human is homozygous for the 1484G->T mutation in the CFTR gene, or (ii) as being a cystic fibrosis carrier when the human is heterozygous for the 1484G->T mutation in the CFTR gene, or (iii) as having no predisposition or carrier status caused by the 1484G->T mutation when the 1484G->T mutation is absent from both alleles of the CFTR gene.
 2. A method of determining if a human is predisposed to cystic fibrosis comprising determining if nucleic acid from the human is homozygous for the 1484 G->T mutation in the cystic fibrosis transmembrane regulatory (CFTR) gene and identifying said human as predisposed to cystic fibrosis when the human is homozygous.
 3. A method of identifying a human as having an increased likelihood of having an offspring afflicted with cystic fibrosis, comprising determining if nucleic acid from the human has the 1484 G->T mutation in one or both alleles of the CFTR gene and identifying said human as having an increased likelihood of having an offspring afflicted with cystic fibrosis when said 1484 G->T mutation is present in at least one allele.
 4. The method of any one of claims 1-2, and 3 wherein genomic DNA is tested for the presence of said mutation.
 5. The method of any one of claims 1-2, and 3 wherein a cDNA copy of the CFTR coding sequence is tested for the presence of said mutation.
 6. The method of any one of claims 1-2, and 3 wherein sequence from the CFTR gene is amplified by the polymerase chain reaction and the amplified sequence is tested for the presence of said mutation.
 7. The method of any one of claims 1-2, and 3 wherein the presence of said mutation is determined by nucleic acid sequencing.
 8. The method of any one of claims 1-2, and 3 wherein the presence of said mutation is determined by restriction fragment length polymorphism analysis following treatment of CFTR gene sequence with at least one endonuclease restriction enzyme.
 9. The method of any one of claims 1-2, and 3 wherein the presence of said mutation is determined by allele specific amplification.
 10. The method of any one of claims 1-2, and 3 wherein the presence of said mutation is determined by primer extension.
 11. The method of any one of claims 1-2, and 3 wherein the presence of said mutation is determined by oligonucleotide ligation.
 12. The method of any one of claims 1-2, and 3 wherein the presence of said mutation is determined by hybridization with a detectably labeled probe.
 13. The method of any one of claims 1-2, and 3 wherein said nucleic acid is further assessed for the presence of one or more mutations selected from the group consisting of 605G->C, 1198-1203del/1204G->A (deletes TGGGCT and replaces G with A at position 1204), 1573A->G, 1604G->C, 1641-1642AG->T, 2949-2953del (deletes TACTC), 2978A->T, 3239C->A, and 3429C->A. 